Jennings Martin D, Kershaw Christopher J, Adomavicius Tomas, Pavitt Graham D
Division of Molecular and Cellular Function, Faculty of Biology Medicine and Health, The University of Manchester, Manchester, United Kingdom.
Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.
Elife. 2017 Mar 18;6:e24542. doi: 10.7554/eLife.24542.
Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B.
真核起始因子2α（eIF2α）的磷酸化通过限制活性eIF2 - GTP/甲硫氨酰 - tRNAi三元复合物（TC）的水平来控制翻译起始。这调节了所有真核mRNA的表达，并参与细胞整合应激反应。控制eIF2活性的关键是翻译因子eIF2B和eIF5，它们分别主要与eIF2 - GDP和TC相互作用。我们采用纯化蛋白的稳态动力学方法证明，无论是否存在竞争性鸟嘌呤核苷酸，eIF2B都以相同的亲和力与eIF2结合。我们发现eIF2B可以与甲硫氨酰 - tRNAi竞争eIF2 - GTP，并能使TC不稳定。当TC由未磷酸化的eIF2形成时，eIF5可以胜过eIF2B以稳定TC/eIF5复合物。然而，当TC/eIF5由磷酸化的eIF2形成时，eIF2B胜过eIF5并使TC不稳定。这些数据揭示了eIF2B和eIF5对TC的竞争，并确定磷酸化的eIF2 - GTP翻译起始中间复合物可被eIF2B抑制。
